The loss of immunogenic epitopes by tumors has urged the development of vaccines against multiple epitopes. Recombinant DNA technologies have opened the possibility to develop multiepitope vaccines in a relatively rapid and efficient way. In this study, several DNA fragments encoding multiple cytotoxic T lymphocyte (CTL) and T helper (Th) cell epitopes were selected from human prostate-specific membrane antigen (hPSM), mouse prostatic acid phosphatase (mPAP), and human prostate-specific antigen (hPSA), These DNA fragments were ligated together to form a novel fusion gene, termed 3P gene. The 3P gene and human IgG Fc gene were inserted into pcDNA3.1 to construct a DNA vaccine designated psig-3P-Fc. Vaccination with psig-3P-Fc by gene gun inoculation induced strong antitumor response in a mouse tumor model, which significantly inhibited tumor growth and prolonged survival time of the tumor-bearing mice. In vitro, when lymphocytes were stimulated by psig-3P-Fc-transfected autologous peripheral blood mononuclear cells (PBMC), CTLs were induced which could specifically kill hPSM-, hPAP-, or hPSA-expressing tumor cells. These observations provide a new vaccine strategy for cancer therapy through concomitant enhancement of antigen specific CD4(+) helper and CD8(+) cytotoxic T-cell responses against tumors.